Plasma proteins from several components of the immune system differentiate chronic widespread pain patients from healthy controls – an exploratory case-control study combining targeted and non-targeted protein identification

1. Introduction

Chronic widespread pain (CWP), including fibromyalgia (FM), has a high population prevalence (5%–10%) with a female predominance.[1–3] CWP is often associated with depressive and anxiety symptoms (e.g., insomnia) as well as their consequences such as personal suffering and high socioeconomic costs.[4–6] Nociplastic pain is a new pain mechanistic descriptor (IASP definition: “Pain that arises from altered nociception despite no clear evidence of actual or threatened tissue damage causing the activation of peripheral nociceptors or evidence for disease or lesion of the somatosensory system causing the pain.” Source: https://www.iasp-pain.org/Education/Content.aspx?ItemNumber=1698#Nociplasticpain), which classifies FM (a subgroup of CWP) as a nociplastic pain condition.[7]

Clinical management, including interventions, are hampered by lack of valid biomarkers.[8,9] Omic methods are increasingly applied in pain research as emerging insights indicate that pain involves complex patterns of molecular changes.[10] The proteome of a tissue regulates biological processes and integrates the effects of genes with, for example, age, behaviors, comorbidities, drugs, and environmental factors.[10–12] In 2015, we reported significant differences in the trapezius muscle proteome between CWP (mainly FM) and controls.[13] Later, we and others have found significant differences in plasma/serum proteome between CWP/FM and healthy controls.[14–17] Proteins obtained from proteomic analysis are generally at nano and micro molar levels, whereas inflammation-related substances (e.g., cytokines and chemokines) are typically found at pico molar levels. Also, targeted omics-like methods using panels of 70 to 90 inflammation-related proteins [mainly cytokines and chemokines (Hereafter labeled Inflammatory panel.)] have been applied when investigating CWP/FM; proteins from such panels can significantly differentiate CWP/FM patients from controls.[18,19]

We recently published two studies of the same subjects aimed at differentiating CWP from control (CON) using plasma biomarkers.[14,18] In the first study, proteomic analyses (two-dimensional gel electrophoresis [2-DE]: number of proteins: 325) were applied and a highly significant differentiation was obtained with 22 significant proteins (e.g., acute phase proteins, complement factors, and coagulation factors) (R2 = 0.84, Q2 = 0.60).[14] In the second study, an inflammation panel of 92 proteins was used, and 11 proteins (e.g., 3 chemokines, 1 cytokine, and 2 growth factors) significantly differentiated the two groups (R2 = 0.58, Q2 = 0.37). Thus, proteins identified using proteomic analysis were somewhat more important than the proteins from the inflammatory panel for group differentiation. As the important proteins identified in the two studies are mainly part of the host defence/immunity system, it is reasonable to assume tight interactions between the different components of this system. However, the relative importance of the proteins from these two protein sources for group differentiation is not known. Understanding this may be important for the design of larger biomarker studies of CWP/FM and in the long perspective for management and treatment of CWP/FM.

Based on the two previous studies,[14,18] we assumed that it will be possible to obtain a significant regression differentiating the two groups of subjects using a broad pattern of plasma proteins from pico to micro molar levels. Hence, this exploratory study investigates the relative importance of proteins obtained from the two sources—i.e., a targeted inflammatory panel and proteins obtained from proteomics (untargeted analysis)–for group differentiation (CWP and CON) and whether a significant protein-protein association network exists among the important proteins. In addition, this exploratory study maps the biological processes involved according to the gene ontology (GO).

2. Subjects and Methods 2.1. Subjects

The two groups of subjects have been described in detail elsewhere.[20] All subjects received a standardized clinical examination to confirm the individual eligibility. As this project was initiated in 2009, the American College of Rheumatology (ACR) criteria from 1990 was used to classify FM/CWP.[6] None of the subjects used any anticoagulatory, opioid, or steroidal medication. Exclusion criteria were medical history record of bursitis, tendonitis, capsulitis, postoperative conditions in the neck/shoulder area, previous neck trauma, disorder of the spine, neurological disease, rheumatoid arthritis or any other systemic diseases, metabolic disease, malignancy, severe psychiatric illness, pregnancy, and difficulties understanding Swedish. A total of 19 CWP and 24 CON were initially recruited, and, as previously reported, 15 CWP patients and 23 CON subjects were included in the final cohorts[18,21]; for a flow chart see.[22]

The study was approved by the Regional Ethical Review Board in Linköping, Sweden (Dnr. M10–08, M233–09, Dnr. 2010/164–32) and followed the guidelines in the Declaration of Helsinki. All participants signed a written consent before the start of the study and after receiving verbal and written information about the objectives and procedures of the study.

2.2. Clinical variables 2.2.1. Age and body mass index.

Age (years), weight (kg), and height (m) were registered at the clinical examination. Body mass index (BMI) was calculated as weight/height2 (kg/m2). All subjects answered a brief health questionnaire and these data have been presented elsewhere[21,23] and here some of these variables (Patient Reported Outcome Measures [PROM]) are used as brief clinical descriptions of the two groups (Table 1). Descriptions of these PROM measures are given below; for details, see.[21,23]

Table 1 - Background and PROM variables (Mean and SD together with Median and Range) for CON and CWP; data have been published elsewhere.[21,23] Group CON n = 23 CWP n = 15 Statistics Variables Mean SD Median Range Mean SD Median Range P value Age (yr) 41.0 10.2 42.0 29.0 49.2 8.9 50.0 31.0 .014 BMI (kg/m2) 24.0 2.8 23.2 12.4 26.0 5.0 23.8 15.6 .185 NRS-whole body 0.0 0.0 0.0 0.0 4.9 2.0 6.0 7.0 <.001 HADS 3.3 2.8 2.0 9.0 14.0 5.3 13.0 17.0 <.001 QOLS 93.1 9.7 94.0 37.0 82.5 13.1 85.0 46.0 .013

BMI = body mass index, CON = healthy control group, CWP = chronic widespread pain group, HADS = Hospital Anxiety and Depression Scale, NRS = Numeric Rating Scale, PROM = Patient Reported Outcome Measures, QOLS = Quality-of-Life Scale.


2.2.2. Pain intensity.

The subjects rated their pain intensity on the whole body using an 11-grade (0–10) Numeric Rating Scale with two endpoints: zero indicating no pain at all and 10 indicating worst possible pain.[24]

2.2.3. Psychological distress.

The Hospital Anxiety and Depression Scale measures symptoms of anxiety and depression using 14 items.[25] A total score of Hospital Anxiety and Depression Scale (score range: 0–42) was used to indicate psychological distress.[26]

2.2.4. Quality-of-Life.

The Quality-of-Life Scale is composed by 16 items measured on seven-point satisfaction scales (total score: 16–112).[27] A higher total score reflects higher satisfaction.

2.3. Protein analyses

Venous blood samples were collected in a EDTA vacutainer and centrifuged (1000 × g, 15 minutes) and plasma was collected. 2-DE in combination with mass spectrometry (MALDI-TOF MS and nLC-MS/MS) was used for the proteomic analysis as described previously.[13,14,28] Briefly, depleted plasma proteins were separated first according to pI (isoelectric focusing) in the first dimension then according to the molecular weight in the second dimension. The separated proteins were stained fluorescently by SYPRO Ruby® (Bio-Rad Laboratories, Hercules, CA) and visualized/analyzed as digitizing images using a charge-coupled device camera (VersaDoc™ Imaging system 4000 MP; Bio-Rad) in combination with a software system designed for evaluation of 2-DE and quantification of protein spots (PDQuest Advanced v. 8.0.1, Bio-Rad). The protein spots of interest were excised from the gels. After enzymatic digestion by trypsin, the samples were subjected to mass spectrometer. A database search of proteins was used to identify the obtained peptide peak list from mass spectrometer as previously described.[14,29]

A multiplex proximity extension assay (PEA) was used to analyze a panel (n = 92) of inflammation related proteins (mainly cytokines, chemokines, and growth factors; see Supplementary Digital Content text file #1, https://links.lww.com/MD/H551). This targeted PEA was conducted using Proseek® Multiplex Inflammation I (Olink Bioscience, Uppsala, Sweden); for details, see previous publication.[18]

2.4. Statistics

For comparison, background and PROM variables are reported mean ± SD together with median and range. For group comparisons of values of background and PROM variables, Student’s t test was applied using IBM SPSS (version 27.0; IBM Corporation, Route 100 Somers, New York, NY). P < .05 was considered significant in all analyses. Analysing omics data and a large number of variables in relation to number of subjects (i.e., broad, and short data tables) requires advanced statistical techniques such as Multivariate Data Analysis (MVDA).[30,31] Here, we have use the recommendations for MVDA of omics data.[30] We have investigated which proteins can be used to differentiate CWP from CON (i.e., group belonging) with Orthogonal Partial Least Squares discriminant analysis (OPLS-DA) using SIMCA-P + (version 17.0; Sartorius Stedim Biotech, Umeå, Sweden).[32] As this procedure has been described in detail elsewhere,[13,14,23,29] we only give a summary. All variables were mean centered, scaled to unit variance (UV-scaling), and log-transformed if necessary. No multivariate outliers were identified according to a principal component analysis. Variables with variable influence on projection (predictive) value (VIPpred) > 1.0 (combined with jack-knifed 95% confidence intervals in the regression coefficients plot not including zero) and with absolute p(corr) ≥ 0.40 were considered significant; p(corr) is the loading of each variable scaled as a correlation coefficient and therefore results in a standardized range from −1 to +1.[30] Number of predictive and orthogonal components are also reported. The OPLS-DA was made in two steps. In the first step, all identified proteins from 2-DE and inflammatory panel analysis (several hundred) were included in the analysis. In the second step, the proteins with VIPpred > 1.5 were used in a new OPLS-DA provided that the first analysis resulted in a significant component according to the internal rules used in SIMCA-P+.[32] When investigating the possible influences of age and BMI, we added the two variables to the final OPLS-DA regression of group membership and analyzed whether they were significant. Furthermore, an OPLS regression was made using the t score of the predictive component of the second OPLS-DA as a dependent variable and with age and BMI as independent variables. R2 describes the goodness of fit and Q2 describes goodness of prediction.[32] Cross validated analysis of variance (CV-ANOVA) with a P ≤ .05 was used to validate the obtained models.

2.5. Network analysis

The protein-protein association network of the important proteins for group differentiation was analyzed using the online database Search Tool for Retrieval of Interacting Genes/Proteins (STRING; version 11).[33] Protein accession numbers (UniProt) for the important significant proteins (i.e., proteins with VIPpred ≥ 1.5) were entered in STRING’s search engine (multiple proteins) with the following parameters: species set to Homo sapiens; maximum number of interactions for query proteins only; and interaction score set to minimum required interaction score of medium confidence (0.400). For the obtained network, PPI enrichment P value and average local clustering coefficient were reported. In the network figure, each protein is represented by a coloured node, and protein-protein interaction and association are represented by an edge visualized as a line. Higher combined confidence scores are represented by thicker lines/edges. Using Protein ANalysis THrough Evolutionary Relationships (PANTHER; version 16.0), the Cellular Component, Molecular Function, and Biological Processes according to the Gene Ontology (GO; http://geneontology.org/docs/ontology-documentation/) were listed.[34] The following settings were used in PANTHER: Organism: Homo sapiens; Analysis: complete; Test type: Fisher’s Exact; and Correction: Bonferroni correction for multiple testing.

3. Results 3.1. Clinical variables

The two groups have been presented earlier and some clinical measures are reported in Table 1.[21,23] Hence, the CON group was younger, had a higher quality of life, and had significantly lower levels of psychological distress. The CWP group had considerable pain intensities, whereas the CON group was pain free.

3.2. Regression analysis of group belonging

Using all proteins (proteomic analysis: n = 325; measurable proteins from the inflammation panel: n = 72), we obtained a significant OPLS-DA with the following characteristics: R2 = 0.996; Q2 = 0.529; and CV-ANOVA P value = .0044 (one predictive and three orthogonal components). When only the most important proteins were included (i.e., VIPpred > 1.5) (Table 2), the significant regression had the following characteristics: R2 = 0.829; Q2 = 0.644; CV-ANOVA P value = 7.0e-07 (one predictive and one orthogonal component). In total, 58 proteins (including proteoforms) had a VIPpred > 1.5.

Table 2 - OPLS-DA regression of group membership (CWP or CON). The most important plasma proteins (i.e., VIPpred > 1.5) are presented in alphabetical order. Numbers of proteoforms are reported together with mean VIPpred and mean p(corr). A positive p(corr) indicates higher levels in CWP. Characterisation is in a pain-immune-nociception context. Proteins in bold are from the panel of 92 inflammation-related proteins. Accession number Protein Abbreviation No. of proteoforms Mean VIPpred Mean p(corr) Characterisation Q13541 Eukaryotic translation initiation factor 4E-binding protein 1 EIF4EBP1/4E-BP1 1 1.59 0.46 Control of protein translation & sensitivity of neurons P01009 Alpha-1-antitrypsin SERPINA1 2 1.62 0.43 Acute phase protein P08697 Alpha-2-antiplasmin SERPINF2 2 1.60 0.43 Acute phase protein P02765 Alpha-2-HS-glycoprotein AHSG 1 2.33 0.62 Immune response P01023 Alpha-2-macroglobulin A2M 2 1.55 0.41 Acute phase protein P02647 Apolipoprotein A-I APOA1 2 1.69 −0.45 Antiinflammation O15169 AXIN1 AXIN1 1 2.05 0.58 Downregulates Wnt signaling pathway Q14790 Caspase-8 CASP8 1 1.51 0.43 Modulate IL-1b & inflammation; Regulation of NF-κB P78556 C-C motif chemokine 20 CCL20 1 1.94 0.53 Chemokine P00450 Ceruloplasmin CP 10 1.85 0.49 Acute phase protein P05160 Coagulation factor XIII B chain F13B 1 1.77 0.47 Coagulation factor P00736 Complement C1r subcomponent C1R 7 1.90 0.51 Complement factors P01024 Complement C3c alpha chain fragment 2 C3 1 2.42 0.64 Complement factors P00751 Complement factor B CFB 1 1.66 0.44 Complement factors P05156 Complement factor I CFI 1 2.42 0.64 Complement factors P02679 Fibrinogen gamma chain FGG 3 1.93 0.51 Acute phase protein O75636 Ficolin-3 FCN3 1 1.75 0.47 Pattern recognition molecule—activate complement P06396 Gelsolin GSN 2 1.75 0.47 Anti-inflammatory P00738 Haptoglobin HP 1 1.73 0.46 Acute phase protein P02790 Hemopexin HPX 1 1.50 0.40 Acute phase protein P01877 Ig alpha-2 chain C region IGHA2 1 1.73 −0.46 Immunoglobulin P42702 Leukaemia inhibitory factor receptor LIFR 1 1.56 −0.39 Activates JAK/STAT and MAPK signaling pathways P00747 Plasminogen PLG 5 1.87 0.50 Acute phase protein P02787 Serotransferrin TF 1 1.77 −0.47 Acute phase protein Q8IXJ6 NAD-dependent protein deacetylase sirtuin-2 SIRT2 1 1.74 0.50 Anti-inflammatory; Regulation of NF-κB O95630 STAM-binding protein STAMPB 1 1.62 0.47 regulation of endosomal-lysosomal degradation pathway P01135 Protransforming growth factor alpha TGFA 1 1.59 0.46 Restoring function in neurodegenerative disorders P02766 Transthyretin TTR 1 1.79 −0.48 Acute phase protein O43508 Tumour necrosis factor ligand superfamily member 12 TNFSF12 1 1.65 0.44 Cytokine P02774 Vitamin D-binding protein GC 3 1.78 0.47 Immunity

AXIN1 = axin-1, CON = healthy control group, CWP = chronic widespread pain group, GC = vitamin d-binding protein, IL = interleukin, JAK/STAT = Janus kinase-signal transducer and activator of transcription pathway, MAPK = mitogen-activated protein kinase signaling pathway, NF-κB = nuclear factor kappa-light-chain-enhancer of activated b cells pathway, OPLS-DA = Orthogonal Partial Least Squares discriminant analysis, VIPpred = variable influence on projection (predictive) value, TF = serotransferrin, TGFA = protransforming growth factor alpha, TNFSF12 = tumour necrosis factor ligand superfamily member 12.

Both proteins determined from the 2-DE analysis and from the inflammation panel were important for the group differentiation although the majority belonged to the proteins from the proteome (Table 2). The four most important proteins for the group differentiation (i.e., highest absolute p(corr) and VIPpred value) were two complement factors (Complement C3c alpha chain fragment 2 and Complement factor I), alpha-2-HS-glycoprotein, and AXIN1.

Several of the important proteins had proteoforms; most proteoforms were noted for ceruloplasmin (n = 10), complement C1r subcomponent (n = 7), and plasminogen (n = 5).

Most proteins were increased in CWP (i.e., a positive p(corr)) but apoliprotein A-I, Ig alpha-2 chain C region, leukaemia inhibitory factor receptor [LIFR], serotransferrin, and transthyretin were higher in the CON (i.e., negative p(corr)) (Table 2).

3.3. Possible influences from age and BMI

When age and BMI were added as regressors to the second OPLS-DA regression of group membership, none of these two variables were significant regressors (i.e., VIPpred < 1.0). We also regressed the t score of the predictive component obtained from the second OPLS-DA using age and BMI as regressors, but the model was not significant. Hence, age and BMI did not significantly influence the important proteins differentiating the CWP from the CON.

3.4. The network and enrichment analysis together with GO analysis

The network and enrichment analysis of the important proteins differentiating CWP from CON (n = 58) (Table 2) identified a significantly enriched protein-protein interaction network (note that proteoforms cannot be included in STRING) (Fig. 1). This means that the proteins included in the analysis have more interactions among themselves than what would be expected for a random set of proteins of similar size drawn from the genome. Thus, the proteins are at least partially biologically connected as a group. When scrutinizing Figure 1, it was found that five of nine proteins from the inflammation panel did not show interactions: SIRT2, AXIN1, LIFR, EIF4EBP1 (4E-BP1), and STAMPB.

F1Figure 1.:

Network analyses of important proteins differentiating CWP from CON. The network had the following characteristics: number of nodes: 29; number of edges: 129; average node degree: 8.9; average local clustering coefficient: 0.62; expected number of edges: 10; PPI enrichment P value: < 1.0e-16. Note that Ig alpha-2 chain C region is not included in STRING. A2M = alpha-2-macroglobulin, AHSG = alpha-2-HS-glycoprotein, APOA1 = apolipoprotein A-I, AXIN1 = axin-1, C1R = complement C1r subcomponent, C3 = complement C3, CASP8 = caspase-8, CCL20 = C-C motif chemokine 20, CFB = complement factor B, CFI = complement factor I, CP = ceruloplasmin, EIF4EBP1 = eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), F13B = coagulation factor XIII B chain, FCN3 = ficolin-3, FGG = fibrinogen gamma chain, GC = vitamin D-binding protein, GSN = gelsolin, HP = haptoglobin, HPX = hemopexin, LIFR = leukaemia inhibitory factor receptor, PLG = plasminogen, SERPINA1 = alpha-1-antitrypsin, SERPINF2 = alpha-2-antiplasmin, SIRT2 = NAD-dependent protein deacetylase sirtuin-2, STAMBP = STAM-binding protein, TF = serotransferrin, TGFA = protransforming growth factor alpha, TNFSF12 = tumour necrosis factor ligand superfamily member 12, TTR = transthyretin.

Biological Processes according to the GO analysis (PANTHER) identified in the enriched network included acute phase response, regulation of complement activation, complement activation, regulation of endopeptidase activity, fibrinolysis, and innate immune response (see Supplementary Digital Content Table 1, https://links.lww.com/MD/H552). Detailed results from the GO analyses are presented in the Supplementary Digital Content Tables 1–3, https://links.lww.com/MD/H552: https://links.lww.com/MD/H553: https://links.lww.com/MD/H554; for molecular function and cellular component see Supplementary Digital Content Tables 2–3, https://links.lww.com/MD/H553: https://links.lww.com/MD/H554.

4. Discussion

As expected, a broad pattern of mainly interacting immune- and inflammation-related plasma proteins from pico to micro molar levels differentiated CWP from CON. Proteins from the plasma proteomics were somewhat more important than proteins from the inflammation panel. The pattern of interacting proteins was neither age- nor BMI-dependent. The protein-protein interaction network was significantly enriched, and the identified biological processes included acute phase response, aspects of complement activation, and innate immune response. The broad involvement of the immune system peripherally supports suggestions that systemic low-grade inflammation is present in CWP.

Both proteins identified from the proteome and from the inflammatory panel contribute to group differentiation (CWP vs. CON). Most of the important proteins were obtained from the plasma proteome although proteins from the inflammation panel also contributed (Table 2). Our results broaden the perspective and clearly suggest that proteins from pico to micro molar levels must be cons

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